Abstract Multi-drug resistance (MDR) in Gram-negative pathogens, including Pseudomonas aeruginosa, poses a significant threat for effective treatment of infections caused by these organisms. A major component in the development of the MDR phenotype in P. aeruginosa is overexpression of the Resistance Nodulation Development (RND)-family efflux pumps, which pump antibacterial agents and biocides out of the cell, and are involved in virulence, biofilm formation, and acquired antibiotic resistance. The overall goal of this proposal is to identify potent inhibitors of the RND-family efflux pumps in P. aeruginosa and develop them into innovative therapeutics, which will be used to increase the potency of existing and new antibiotics and decrease the emergence of MDR bacteria. Our strategy is to use an innovative cellular bioluminescent reporter assay to screen a library of small molecules and natural products for efflux pump inhibitors (EPIs). In preliminary studies, we developed a high throughput screen (HTS) for fatty acid synthesis II (FASII) inhibitors using a PfabD- luxCDABE reporter strain, which induces a strong and reproducible luminescent signal in response to FASII inhibitors in an efflux-deficient, but not an efflux-proficient strain, indicating the FASII inhibitors are RND pump substrates. Therefore, the efflux proficient PfabD-luxCDABE reporter strain grown in the presence of a FASII inhibitor provides a sensitive, gain-of signal reporter assay to screen for inhibitors of RND efflux pumps, which we verified using a known EPI (PA?N). In Phase I, we will optimize the PfabD-luxCDABE cell-based reporter assay for high-throughput screening and we will validate the assay in a small-scale pilot screen of known bioactive compounds. The validated assay will be applied to screen a library of ?350,000 compounds at Microbiotix and at the ICCB (Harvard Medical School). Primary hit compounds will be confirmed, tested against a counter-screen to eliminate non-specific inhibitors, and prioritized based on their potency and medicinal chemistry properties. The high priority compounds will be evaluated in a panel of secondary assays that will assess EPI activity, spectrum of activity, cytotoxicity, selectivity, solubility, and liver microsome stability. We will order commercially available analogs, as available, of high priority hits to establish preliminary structure activity relationships (SAR). We expect to identify 3-5 validated hits that meet the stringent criteria described in the milestones of each specific aim. These compounds will be optimized in Phase II to generate Lead compounds for drug development. To achieve the goal of this Phase I proposal we will complete the following Specific Aims: Aim 1. Develop and optimize a cell-based reporter assay for efflux pump inhibitors in P. aeruginosa. Aim 2. Screen a diverse small molecule library for compounds that are potent inhibitors of efflux pumps in P. aeruginosa. Aim 3. Validate confirmed hits using secondary assays and identify hit-to-lead series.